Lab 10 Protein Extractions

enzymes

thousands, rubisco, alcohol dehydrogenase

structural proteins

provides mechanical support

ex: collagen

motor proteins

protein function, generate movement, myosin, flagellin

receptor proteins

protein function, rhodopsin in retina, insulin receptor

storage

protein function, casein, endosperm

transport

protein function, carry ions or small molecules

ex: serum albumin

regulation

protein function, DNA binding proteins, transcriptional activators

signaling

protein function, stress response hormones, growth factors, EGF

special purpose proteins

protein function, selective advantage, highly variable

ex: antifreeze proteins, GFP

proteins determine..

cell structure, cell function, each has unique AA sequence

unique sequence determines..

how polypeptide chain will fold to form molecule w/ distinctive shape and chemistry

What covalently holds AA together?

peptide bonds

what are proteins made of?

long chain of unique combination of AA

how do they fold?

conformation of lowest energy. fold determines functions/properties

denaturation

change in chemical structural and biological properties of proteins leads to denaturation

loss of secondary, tertiary, quaternary, but primary stays unaffected

what leads to protein denaturation?

extreme temp, pH change, sonication or mechanical shearing, chemicals, salt conc., solvents, alcohols, other chemical substances (like SDS)

effects of protein denaturation

loss of stability, function

effects of loss of stability

unfolding, permanent changes in structure and folding proteins

effects of loss of function

most enzymes, changes in binding sites, substrate specificity

renaturation

regain activity, globular proteins

characteristics of good buffers

low metal binding capabilities, free of side effects, protective chemicals, maintaining native structure of proteins

ex: Tris, phosphate

thiol compound (common addition to extraction buffers)

frequently added to protect proteins from oxidation

ex: DTT or beta-ME

Chelating agents (common addition to extraction buffers)

protect enzymes from inactivation by heavy metals ions

prevents protein-metal ion aggregation/precipitation, substrate inhibition, or proteolysis

ex: EDTA

cations (common addition to extraction buffers)

maintain ionic strength (K+, Na+) or provide specific stabilizing interactions (Mg+2)

substrates (common addition to extraction buffers)

added to stabilize enzymes, specific

protease inhibitors (common addition to extraction buffers)

suppression of endogenous proteases

ex: PMSF inactivates # of serine proteases

osmotically active solutes (common addition to extraction buffers)

maintains tonicity of solution comparable to cell or tissue. avoids swelling or plasmolyzing of plastids and mitochondria

ex: glycerol / other polyols to stabilize enzymes by increasing solution viscosity

detergents (common addition to extraction buffers)

added to solubilize organelles or membrane associated proteins

ex: triton X-100, nonionic detergent

PVPP (common addition to extraction buffers)

added (2-10% w/w) to plant extracts to prevent browning from alkaloids and polyphenolic compounds

Protein Quantification Methods: UV absorbance

simple, error prone

Protein Quantification Methods: Biuret assay

compatible with most surfactants, linear response curve, less protein-protein variation than Coomassie dye, BUT incompatible w/ substance that reduce copper, incompatible w/ common reducing agents

ex: BCA assay and Lowry assay

Protein Quantification Methods: Calorimetric dye-based assay

protein-dye binding and direct detection of color change, fast and easy, compatible w/ most salts/solvents/buffers/thiols/reducing substances/metal-chelating agents, incompatible w surfactants/protein-protein variation

ex: Bradford, Coomassie based

Protein Quantification Methods: Fluorescent dye

protein-dye binding and direct detection of increase in fluorescence associated with the bound dye

excellent sensitivity, requiring less protein sample for quantitation, can be adapted for automated handling in high-throughout applications, BUT requires specialized tools

Bradford Assay

calorimetric method, coomassie dye-binding assay, fast and simple, compatible w/ buffer salts/metal ions/reducing agents/chelators, less tolerant to detergents, RT, simple tools, detects protein conc at 2-1500 ug/mL range

Coomassie dye color change

when acidic, reddish-brown to blue (595 nm)